U.S. patent application number 10/297301 was filed with the patent office on 2003-09-25 for nucleic acid isolation.
Invention is credited to Andreassen, Jack, Bergholtz, Stine, Korsnes, Lars.
Application Number | 20030180754 10/297301 |
Document ID | / |
Family ID | 9893009 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030180754 |
Kind Code |
A1 |
Bergholtz, Stine ; et
al. |
September 25, 2003 |
Nucleic acid isolation
Abstract
The present invention relates to a method of isolating nucleic
acid from a blood sample, said method comprising: (a) selectively
isolating leucocytes from said sample by binding said leucocytes to
a solid support by means of a binding partner specific for
leucocytes; (b) lysing said isolated leucocytes; and (c) binding
nucleic acid released from said lysed cells to said solid support.
Kits for isolating nucleic acid from samples form further
embodiments of the invention.
Inventors: |
Bergholtz, Stine; (Oslo,
NO) ; Korsnes, Lars; (Oslo, NO) ; Andreassen,
Jack; (Oslo, NO) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Family ID: |
9893009 |
Appl. No.: |
10/297301 |
Filed: |
April 30, 2003 |
PCT Filed: |
June 5, 2001 |
PCT NO: |
PCT/GB01/02472 |
Current U.S.
Class: |
506/3 ; 435/270;
435/6.11; 435/7.2 |
Current CPC
Class: |
C07H 21/00 20130101;
C07B 2200/11 20130101; C12N 15/1006 20130101 |
Class at
Publication: |
435/6 ; 435/7.2;
435/270 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/567; C12N 001/08 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 5, 2000 |
GB |
0013658.0 |
Claims
1. A method of isolating nucleic acid from a blood sample, said
method comprising: (a) selectively isolating leucocytes from said
sample by binding said leucocytes to a solid support by means of a
binding partner specific for leucocytes; (b) lysing said isolated
leucocytes; and (c) binding nucleic acid released from said lysed
cells to said solid support.
2. The method of claim 1 wherein in step (a) leucocytes in said
sample are bound to a leucocyte-specific binding partner, said
binding partner being attached to a solid support before or after
binding to said leucocytes, thereby to bind said support to said
leucocytes.
3. The method of claim 1 or claim 2 wherein the nucleic acid is
DNA, RNA or any naturally occurring modification thereof, or
combinations thereof.
4. The method as claimed in any one of claims 1 to 3 wherein the
binding partner in step (a) binds specifically to leucocytes
present in the sample but not to other cells or components of the
sample.
5. The method of claim 4 wherein said binding partner is an
antibody or a fragment or derivative of an antibody.
6. The method of any one of claims 1 to 5 wherein one or more
different binding partners are used to isolate the leucocytes.
7. The method of any one of claims 1 to 6 wherein all or
substantially all leucocytes present in the sample are
separated.
8. The method of any one of claims 1 to 7 wherein the binding
partner recognises or is capable of binding specifically to one or
more of the molecules selected from the group comprising HLA-I,
CD11a, CD18, CD45, CD46, CD50, CD82, CD100, CD162, CD5 and CD
15.
9. The method of claim 8 wherein one or more of said binding
partners are used.
10. The method of claims 8 or claim 9 wherein a combination
of-binding partners specific for CD45 and CD15 are used.
11. The method of any one of claims 1 to 10 wherein the solid
support of step (a) comprises particles, preferably polymeric
particles.
12. The method of any one of claims 1 to 11 wherein said solid
support is magnetic, preferably superparamagnetic.
13. The method of any one of the preceding claims wherein the
binding partner is attached directly or indirectly to the solid
support of step (a).
14. The method of any one of claims 9 to 13 wherein more than one
different type of binding partner is used and said binding partners
are attached to the same or different solid support.
15. The method of any one of claims 1 to 14 wherein the nucleic
acid binding in step (c) is carried out using a detergent based
system.
16. The method of any one of claims 1 to 15 wherein said isolated
cells of step (b) or the nucleic acid of step (c) are additionally
contacted with an additional amount of a second solid support,
preferably binding said nucleic acid to said second solid support
which is capable of binding nucleic acid.
17. A method of isolating nucleic acid from a cell sample, said
method comprising; (a) selectively isolating cells from said sample
by binding said cells to a solid support by means of one or more
appropriate binding partners specific for said cells; (b) lysing
said isolated cells; (c) binding nucleic acid released from said
lysed cells to said first solid support; and (d) contacting said
isolated cells of step (b) or the nucleic acid of step (c) with an
additional amount of a second solid support, preferably binding
said nucleic acid to said second solid support which is capable of
binding nucleic acid.
18. The method of claim 17 wherein the cells are leucocytes.
19. The method of claim 17 or 18 wherein the cell sample is a blood
sample.
20. The method of any one of claims 17 to 19 wherein 30 the
components used in, or features present in steps (a), (b) and (c)
are as defined in any one of the preceding claims.
21. The method of any one of claims 17 to 20 wherein the isolated
cells are lysed in the presence of said second solid support.
22. The method of any one of claims 18 to 21 wherein the first
solid support is as defined in any one of claims 8 to 10 and the
second solid support which is different to said first solid support
is negatively charged.
23. The method of any one of claims 1 to 22 wherein the isolated
nucleic acid is DNA and the method comprises a further step to
isolate RNA from the sample.
24. A kit for isolating nucleic acid from a sample comprising: (a)
a solid support; (b) means for binding leucocytes to said solid
support; (c) means for lysing said cells; and (d) means for binding
nucleic acid released from said lysed cells to said same solid
support.
25. The kit of claim 24 further comprising one or more of: (e) a
second solid support; (f) a proteinase; and (g) means for detecting
nucleic acid.
26. A kit for isolating nucleic acid from a cell sample comprising:
(a) a solid support; (b) means for binding the particular cells
from which it is desired to isolate nucleic acid to said solid
support; (c) means for lysing said cells; (d) means for binding
nucleic acid released from said lysed cells to said same solid
support; and (e) a second solid support.
27. The method of any one of claims 1 to 23 wherein said method is
performed using an automated system for handling of the solid
support during cell lysis, nucleic acid binding and optionally
washing steps.
28. The method of claim 27 wherein said automated system
additionally handles the support during: the cell isolation
stage.
29. The method of claim 27 or claim 28 wherein the Bead
Retriever.TM. apparatus is used.
Description
[0001] The present invention relates to the isolation of nucleic
acid from blood cells, and especially to a method for isolating DNA
or RNA from such cells which combines a solid phase cell isolation
step with a solid phase DNA or RNA isolation step.
[0002] The isolation of nucleic acid is an important step in many
biochemical and diagnostic procedures. For example, the separation
of nucleic acids from the complex mixtures in which they are often
found is frequently necessary before other studies and procedures
e.g. detection, cloning, sequencing, amplification, hybridisation,
cDNA synthesis, studying nucleic acid structure and composition
(e.g. the methylation pattern of DNA) etc. can be undertaken; the
presence of large amounts of cellular or other contaminating
material e.g. proteins or carbohydrates, in such complex mixtures
often impedes many of the reactions and techniques used in
molecular biology. In addition, DNA may contaminate RNA
preparations and vice versa. Thus, methods for the isolation of
nucleic acids from complex mixtures such as cells, tissues etc. are
demanded, not only from the preparative point of view, but also in
the many methods in use today which rely on the identification of
DNA or RNA e.g. diagnosis of microbial infections, forensic
science, tissue and blood typing, genotyping, detection of genetic
variations etc.
[0003] The use of DNA or RNA identification is now widely accepted
as a means of distinguishing between different cells or cell types
or between variants of the same cell type containing DNA mutations.
Thus, HLA typing, which is more commonly carried out by
identification of characteristic surface antigens using antibodies,
may alternatively be effected by identification of the DNA coding
for such antigens. Microbial infection or contamination may be
identified by nucleic acid analysis to detect the target organism,
rather than relying on detecting characterising features of the
cells of the microorganisms e.g. by detecting morphological or
biochemical features. Genetic variations may be identified by
similar means.
[0004] Particularly in the fields of nucleic acid diagnostics,
population studies and genotyping, it is important to obtain high
quality and pure nucleic acid preparations to ensure that further
amplification and/or detection steps are reliably and accurately
carried out.
[0005] Isolation of nucleic acid from blood cells is required for a
number of applications, including for example typing, or for
diagnostic or screening applications for example to detect
mutations or polymorphisms. For such applications large amounts of
pure nucleic acid, particularly genomic DNA, are desirable.
Particularly, it is desirable to obtain such nucleic acid readily
and speedily and to avoid the use of materials which may
contaminate and/or degrade the nucleic acid.
[0006] A range of methods are known for the isolation of nucleic
acids, but generally speaking, these rely on a complex series of
extraction and washing steps and are time consuming and labourious
to perform. Moreover, the use of materials such as alcohols and
other organic solvents, chaotropes and proteinases is often
involved which is disadvantageous since such materials tend to
interfere with many enzymic reactions and other downstream
processing applications.
[0007] Thus, classical methods for the isolation of nucleic acids
from complex starting materials such as blood or blood products or
tissues involves lysis of the biological material by a detergent or
chaotrope, possibly in the presence of protein degrading enzymes,
followed by several extractions with organic solvents e.g. phenol
and/or chloroform, ethanol precipitation, centrifugations and
dialysis of the nucleic acids. Not only are such methods cumbersome
and time consuming to perform, but the relatively large number of
steps required increases the risk of degradation, sample loss or
cross-contamination of samples where several samples are
simultaneously processed. In the case of RNA isolation, the risk of
DNA contamination is relatively high.
[0008] Improvements in methods for isolating nucleic acids have
been made, and more recently, other methods have been proposed
which rely upon the use of a solid phase. In U.S. Pat. No.
5,234,809, for example, is described a method where nucleic acids
are bound to a solid phase in the form of silica particles, in the
presence of a chaotropic agent such as a guanidinium salt, and
thereby separated from the remainder of the sample. WO 91/12079
describes a method whereby nucleic acid is trapped on the surface
of a solid phase by precipitation. Generally speaking, alcohols and
salts are used as precipitants.
[0009] Whilst such methods speed up the nucleic acid separation
process, a need still exists for methods which are quick and simple
to perform, which enable good yields to be obtained without losses,
and in particular which do not require the use of solvents,
alcohols and similar agents. In addition, particularly where large
quantities of nucleic acid are required to be isolated, methods
which are effective for large as well as small volumes of sample
material are desirable.
[0010] Chaotropes require to be used at high molarity, resulting in
viscous solutions which may be difficult to work with, especially
in RNA work. Amplification procedures such as PCR, and other
enzyme-based reactions, are very sensitive to the inhibitory or
otherwise interfering effects of alcohols and other agents.
Moreover, the drying of the nucleic acid pellet which is necessary
following alcohol precipitation and the problems with dissolving
nucleic acids, are also known to lead to artefacts in enzyme-based
procedures such as PCR. Since such procedures are now a mainstay of
molecular biology, there is a need for improved methods of nucleic
acid isolation from blood samples, and particularly for methods
which are quick and simple to perform and which avoid the use of
chaotropic agents or alcohol precipitation. In addition, as it is
sometimes desirable to isolate relatively large amounts of nucleic
acid from blood samples, there is a need for methods which enable
good yields of nucleic acid to be obtained from both large (e.g. 1
ml to 100 ml or more) and small (e.g. up to 1 ml) blood samples.
There is also a need for a method which allows for differentiation
between RNA and DNA and permits a separate isolation of both types
of nucleic acid from the same sample. The present invention seeks
to provide such methods.
[0011] In particular, it has now been found that nucleic acid may
be isolated from a blood or blood-derived sample in a form suitable
for amplification or other downstream processes, by a simple and
easy to perform procedure which involves specifically isolating
nucleic acid-containing cells from the sample onto a solid support,
lysing the isolated support-bound cells and allowing the released
nucleic acid to bind to the same solid support (or alternatively to
bind to a mixture of the same and different solid supports),
whereupon to bind to the nucleic acid may be readily separated from
the sample, e.g. by removal of the support from the sample. The
binding of the nucleic acid is independent of its sequence.
Moreover, by appropriate choice of nucleic acid binding conditions
and/or the nature of the solid support, it can be selected whether
DNA or RNA binds to the support, thereby enabling a selective DNA
or RNA isolation procedure.
[0012] In one aspect, the present invention thus provides a method
of isolating nucleic acid from a blood sample, said method
comprising:
[0013] (a) selectively isolating leucocytes from said sample by
binding said leucocytes to a solid support by means of a binding
partner specific for leucocytes;
[0014] (b) lysing said isolated leucocytes; and
[0015] (c) binding nucleic acid released from said lysed cells to
said solid support.
[0016] More particularly, in step (a), leucocytes in said sample
may be bound to a leucocyte-specific binding partner, said binding
partner being attached to a solid support before or after binding
to said leucocytes, thereby to bind said support to said
leucocytes.
[0017] The nucleic acid may be DNA, RNA or any naturally occurring
modification thereof, and combinations thereof. Preferably however
the nucleic acid will be DNA, which may be single or double
stranded or in any other form, e.g. linear or circular. The method
of the present invention is particularly suited to isolating
genomic DNA.
[0018] The term "leucocyte" is used herein to include any nucleic
acid-containing cell of the blood. Thus, the term "leucocyte"
includes all white blood cells. Such cells may be lymphoid cells
e.g. lymphocytes such as T-cells and B-cells, or natural killer
(NK) cells or myeloid cells e.g. monocytes/macrophages,
granulocytes/neutrophils, eosinophils, basophils/mast cells,
megakaryocytes and erythroid progenitor cells. Dendritic cells
(both myeloid and lymphoid) are also included. All nucleated cells
which may occur in the blood or haemopoietic system are
included.
[0019] The "blood sample" may be any sample derived from blood
which retains cells, for example whole blood or buffy coat. The
sample may be freshly obtained, or stored or treated in any desired
or convenient way, for example by dilution or adding buffer, or
other solutions or solvents, enzyme-containing solutions etc.), as
long as the integrity of the leucocytes within it is maintained
(i.e. as long as the leucocyte surface remains intact). The "blood
sample" may also be any "blood-related" sample, for example a
sample obtained from other haemopoietic tissues such as bone
marrow, or from other tissues/fluids which may contain cells of
haemopoietic origin, e.g. ascites, lymphatic fluid, or cell
suspensions (e.g. single cell suspensions) obtained from any such
tissues or fluids. Thus, the "blood sample" may be any haemopoietic
sample or any sample (e.g. a clinical sample) containing cells of
haemopoietic origin. Thus, alternatively defined, the invention can
be seen to provide a method, as defined above, which is a method of
isolating nucleic acid from a cell sample (e.g. a clinical sample),
and in particular from such a sample containing cells of
haemopoietic origin. Such a cell sample is thus a sample containing
leucocytes.
[0020] Preferably samples are 10 .mu.l to 100 ml in size,
preferably from 200 .mu.l to 10 ml. The method of the invention may
be used for small samples, e.g. less than 1 ml or for larger
samples e.g. at least 2 ml, e.g. more than 5 ml or 10 ml or 50
ml.
[0021] Affinity-based separation or isolation systems for desired
target cells are well known in the art, and rely on the specificity
of a binding partner, specific or selective for the target cell, to
achieve selective isolation of the cell. Such a system is employed
according to the present invention in order to achieve selective
isolation of leucocytes from the sample. Thus, the binding partner
in step (a) may be any moiety having a binding affinity for a
leucocyte, and in particular a selective or specific binding
affinity such that it binds specifically to leucocytes present in
the sample but not to other cells or components of the sample.
[0022] The binding partner may be any molecule or moiety capable of
binding to a leucocyte, but conveniently will be a protein,
polypeptide or peptide. Other moieties or molecules of a different
chemical nature, for example carbohydrates or small organic
molecules may however also be used. Nucleic acid binding partners
e.g. aptamers may also be used.
[0023] The binding partner may bind to molecules or structures on
the surface of the leucocytes, for example to cell surface antigens
which are expressed (e.g. specifically) on the surface of
leucocytes. Alternatively, the binding partner may be a moiety
binding to a cell surface expressed protein e.g. a cell surface
receptor.
[0024] The binding partner may, for example, conveniently be an
antibody specific for a leucocyte surface antigen. Antibody
fragments and derivatives may also be used, according to techniques
well known in the art.
[0025] Antibodies for use as binding partners in methods of the
present invention may be of any species, class or subtype.
Furthermore the antibody may be natural, derivatised or synthetic.
Representative "antibodies" thus include:
[0026] (a) any of the various classes or sub-classes of
immunoglobulin, e.g. IgG, IgA, IgM, IgD or IgE derived from any
animal e.g. any of the animals conventionally used, e.g. sheep,
rabbits, goats, or mice,
[0027] (b) monoclonal or polyclonal antibodies
[0028] (c) intact antibodies or fragments of antibodies, monoclonal
or polyclonal, the fragments being those which contain the binding
region of the antibody, e.g. fragments devoid of the Fc portion
(e.g. Fab, Fab', F(ab').sub.2, Fv), the so called "half molecule"
fragments obtained by reductive cleavage of the disulphide bonds
connecting the heavy chain components in the intact antibody.
[0029] (d) antibodies produced or modified by recombinant DNA or
other synthetic techniques, including monoclonal antibodies,
fragments of antibodies, "humanised antibodies", chimeric
antibodies, or synthetically made or altered antibody-like
structures. Also included are functional derivatives or
"equivalents" of antibodies e.g. single chain antibodies,
CDR-grafted antibodies, minimum recognition unit antibodies
etc.
[0030] Methods for preparing such fragments or derivatives are well
known in the art and widely described in the literature.
[0031] In addition to antibodies or antibody-based molecules, other
types of binding partner may be used, for example peptide or other
molecules, synthetically made and/or selected from display or
combinatorial libraries e.g. phage display. Mention may be made of
aptamers. Other types of leucocyte-specific binding partner may
include affibodies or other synthetic affinity molecules, and
lectins.
[0032] Leucocytes may express or carry a variety of molecules on
their surface which may be recognised by a specific binding
partner. Such molecules may be common to all or most (e.g.
substantially all) leucocytes (so-called "pan-leucocyte") or they
may be carried/expressed by only a subset of leucocytes, for
example particular cell types such as T-cells, B-cells, lymphocytes
in general, monocytes etc. Ideally, binding partners specific for
pan-leucocyte molecules or antigens are used. However, the
invention permits one or more different binding partners to be
used, and hence combinations or mixtures of binding partners may be
used to achieve the desired separation.
[0033] Thus, a binding partner, or combination or mixture of
binding partners, may be selected to achieve a desired separation
or isolation of leucocytes from the sample. Advantageously, all or
substantially all (i.e. close to all) leucocytes present in the
sample may be separated. The separation achievable may be dependent
not only on the binding partner(s) selected, but also on the nature
of the sample, binding conditions etc. Also, biological systems are
by their nature variable, and 100% separation may not always be
achieved, and, indeed, is not necessary according to the present
invention; as in any biological system, some tolerance must be
allowed for. However, in preferred embodiments of the invention at
least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the leucocytes
present in the sample may be separated.
[0034] Leucocytes express on their surface a range of molecules
classified under the "CD" system and also HLA antigens, which may
be used as "targets" for the leucocyte-specific binding partners.
Preferred binding partners according to the invention thus include
those recognising or capable of binding specifically to one or more
of: HLA-I, CD11a, CD18, CD45, CD46, CD50, CD82, CD100 or CD162. CD5
and/or CD15 may also be included in this list. One or more of such
binding partners may be used.
[0035] Other antigens are expressed more selectively, for example
CD5 is expressed by lymphoid cells, including T- and B-cells and NK
cells. CD15 is expressed by monocytes and neutrophils. HLA-I is
expressed by lymphocytes but not granulocytes. Table 1 below shows
other such antigens, and Table 2 shows the typical distribution of
different leucocyte cell types in a blood sample. Appropriate
combinations of binding partners recognising the different antigens
of Table 1 may be selected to enable the desired separation of
leucocytes from a sample, e.g. to isolate the majority of
leucocytes from a sample.
[0036] A combination of binding partners for CD45 and CD15
represents a preferred embodiment according to the present
invention.
1TABLE 1 Different cell surface molecules expressed in the
haematopoietic system Lymphoid cells Myeloid cells T-cell B-cell
and NK Monocyte Neutrophil CD2 CD3 CD3 + CD8 CD5 CD5 CD13 CD13 CD15
CD15 CD43 CD43 CD88 CD88 CD97 CD97 CD101 CD101 CD107a CD107a
Different cell surface molecules expressed on all the leucocytes:
HLA-I HLA-I HLA-I HLA-I CD11a CD11a CD11a CD11a CD18 CD18 CD18 CD18
CD45 CD45 CD45 CD45* CD46 CD46 CD46 CD46 CD50 CD50 CD50 CD50 CD82
CD82 CD82 CD82 CD100 CD100 CD100 CD100 CD162 CD162 CD162 CD162
*CD45 does not have a very high expression on neutrophils
[0037]
2TABLE 2 Distribution of different leucocytes in normal blood
samples Cell type % range Neutrophil 45-76% Eosinophil 2-4%
Basophil 0.5-1% Monocytes 6-10% Lymphocytes 20-35% (T-cells
60-80%)
[0038] Normally a blood sample contains 6.times.10.sup.9
leucocytes/litre (range 2-12.times.10.sup.9)
[0039] The solid support may be any of the well known supports or
matrices which are currently widely used or proposed for
immobilisation, separation etc. These may take the form of
particles, sheets, gels, filters, membranes (e.g. nylon membranes),
fibres, capillaries, needles or microtitre strips, tubes, plates or
wells, etc, combs, pipette tips, micro arrays, chips, or indeed any
solid surface material.
[0040] Conveniently the support may be made of glass, silica,
latex, plastic or any polymeric material. Generally speaking, for
isolation of DNA, the nature of the support is not critical and a
variety of surface materials may be used. The surface of the solid
support may be hydrophobic or hydrophilic. Preferred are materials
presenting a high surface area for binding of the cells, and
subsequently, of the nucleic acid. Such supports will generally
have an irregular surface and may be for example be porous or
particulate e.g. particles, fibres, webs, sinters or sieves.
Particulate materials e.g. beads are generally preferred due to
their greater binding capacity, particularly polymeric
beads/particles.
[0041] Conveniently, a particulate solid support used according to
the invention will comprise spherical beads. The size of the beads
is not critical, but they may for example be of the order of
diameter of at least 1 and preferably at least 2 .mu.m, and have a
maximum diameter of preferably not more than 10 and more preferably
not more than 6 .mu.m. For example, beads of diameter 2.8 .mu.m and
4.5 .mu.m have been shown to work well.
[0042] Monodisperse particles, that is those which are
substantially uniform in size (e.g. size having a diameter standard
deviation of less than 5%) have the advantage that they provide
very uniform reproducibility of reaction. Monodisperse polymer
particles produced by the technique described in U.S. Pat. No.
4,336,173 are especially suitable.
[0043] Non-magnetic polymer beads suitable for use in the method of
the invention are available from Dynal Particles AS (Lillestrm,
Norway; previously Dyno Particles or Dyno Speciality Polymers) as
well as from Qiagen, Amersham Pharmacia Biotech, Serotec, Seradyne,
Merck, Nippon Paint, Chemagen, Promega, Prolabo, Polysciences,
Agowa, Bangs Laboratories and Dyno Particles or Dyno Speciality
Polymers.
[0044] However, to aid manipulation and separation, magnetic beads
are preferred. The term "magnetic" as used herein means that the
support is capable of having a magnetic moment imparted to it when
placed in a magnetic field, and thus is displaceable under the
action of that field. In other words, a support comprising magnetic
particles may readily be removed by magnetic aggregation, which
provides a quick, simple and efficient way of separating the
particles following the cell and nucleic acid binding steps, and is
a far less rigorous method than traditional techniques such as
centrifugation which generate shear forces which may disrupt cells
or degrade nucleic acids.
[0045] Thus, using the method of the invention, the magnetic
particles with cells attached may be removed onto a suitable
surface by application of a magnetic field e.g. using a permanent
magnet. It is usually sufficient to apply a magnet to the side of
the vessel containing the sample mixture to aggregate the particles
to the wall of the vessel and to pour away the remainder of the
sample.
[0046] Especially preferred are superparamagnetic particles for
example those described by Sintef in EP-A-106873, as magnetic
aggregation and clumping of the particles during reaction can be
avoided, thus ensuring uniform nucleic acid extraction. The
well-known magnetic particles sold by Dynal Biotech ASA (Oslo,
Norway, previously Dynal AS) as DYNABEADS, are particularly suited
to use in the present invention.
[0047] Functionalised coated particles for use in the present
invention may be prepared by modification of the beads according to
U.S. Pat. Nos. 4,336,173, 4,459,378 and 4,654,267. Thus, beads, or
other supports, may be prepared having different types of
functionalised surface, for example positively or negatively
charged, hydrophilic or hydrophobic.
[0048] The binding partner(s) may be attached to the solid support
in any convenient way, before or after binding to the leucocytes,
according to techniques well known in the art and described in the
literature. Thus the binding partner may be attached directly or
indirectly to the solid support.
[0049] In a convenient embodiment, the binding partner may be
attached to the support, prior to contact with the sample. Such
attachment may readily be achieved by methods (e.g. coupling
chemistries) well known in the art, and conveniently the binding
partner is bound directly to the solid support, for example by
coating. However it may also be attached via spacer or linker
moieties. The binding partner may be covalently or reversibly
attached according to choice.
[0050] Alternatively, as mentioned above, the binding partner may
first be brought into contact with the sample, to bind to the
leucocytes before being attached to the solid support. In this
case, the solid support may conveniently carry or be provided with
a binding moiety capable of binding to the leucocyte-specific
binding partner. Again, such binding systems are well known in the
art. For example, the solid surface may carry a (secondary)
antibody capable of binding to the anti-leucocyte binding partner
(e.g. a polyclonal anti-species antibody).
[0051] Where more than one different type of binding partner is
used (e.g. anti-CD45 and anti-CD15 antibodies) they may be attached
to the same or different solid supports. Such a system using
different solid supports, is applicable particularly in the case of
a particulate support such as beads. Thus, different binding
partners may be attached to different beads.
[0052] In embodiments where more than one different type of binding
partner is used, appropriate amounts or ratios at which the
different types of binding partner may be used will be readily
determined by a person skilled in the art. For example, in
preferred embodiments of the present invention, where anti CD45 and
anti CD15 antibodies are used, these may be used at any ratio
providing that said ratio allows cells to be isolated. Preferable
ratios are 1:1 and 2:1 ratios of CD45 to CD15.
[0053] As mentioned above, cell separation techniques based on
solid phase affinity binding (e.g. immunomagnetic separation (IMS))
are well known in the art and conditions to achieve this may
readily be determined by the skilled worker in this field. Thus,
for example a solid support carrying anti-leucocyte binding
partner(s) may be brought into contact with the sample. A
particulate solid support may, for example, be added to the sample
contained (e.g. suspended) in an appropriate medium (e.g. a
buffer). The support may then be left in contact with the sample
(e.g. incubated) for a length of time to enable binding to the
cells to occur. Conditions during the step are not critical, and
the sample-support mixture may be incubated at e.g. 4 to 20.degree.
C. for 10 minutes to 2 hours e.g. 20-45 minutes.
[0054] Following cell binding, the isolated or support-bound cells
are lysed to release their nucleic acid. Methods of cell lysis are
well known in the art and widely described in the literature and
any of the known methods may be used. Any of the following methods
could, for example, be used: detergent lysis using e.g. SDS, LiDS
or sarkosyl in appropriate buffers; the use of chaotropes such as
Guanidium hydrochloride (GHCl), Guanidium thiocyanate (GTC), sodium
iodide (NaI), perchlorate etc; mechanical disruption, such as by a
French press, sonication, grinding with glass beads, alumina or in
liquid nitrogen; enzymatic lysis, for example using lysozyme,
proteinases, pronases or cellulases or any of the other lysis
enzymes commercially available; lysis of cells by bacteriophage or
virus infection; freeze drying; osmotic shock; microwave treatment;
temperature treatment; e.g. by heating or boiling, or freezing,
e.g. in dry ice or liquid nitrogen, and thawing; alkaline lysis. As
mentioned above, all such methods are standard lysis techniques and
are well known in the art, and any such method or combination of
methods may be used.
[0055] As mentioned above, the present invention affords the
advantage that the use of agents such as solvents, alcohols and
chaotropes may be avoided. Thus, whilst lysis methods such as those
mentioned above using such agents may be employed, in advantageous
embodiments of the invention the use of such agents is avoided.
[0056] Conveniently, lysis may be achieved according to the present
invention by using detergents. An exemplary suitable lysis agent
thus includes a detergent such as SDS or another alkali metal
alkylsulphate salt, e.g. LiDS, or Sarkosyl or combinations thereof.
The lysis agents may be supplied in simple aqueous solution, or
they may be included in a buffer solution, to form a so-called
"lysis buffer". Any suitable buffer may be used, including for
example Tris, Bicine, Tricine and phosphate buffers. Alternatively
the lysis agents may be added separately. Salts, for example LiCl
and NaCl, may also be included in or added to the lysis buffers. In
particular, LiCl is preferred when LiDS is used and NaCl is
preferred when SDS is used.
[0057] Suitable concentrations and amounts of lysis agents will
vary according to the precise system etc. and may be appropriately
determined, but concentrations of e.g. 2M to 7M chaotropes such as
GTC GHCl, NaI or perchlorate may be used, 0.1M to 1M alkaline
agents such as NaOH, and 0.1 to 50% (w/v) e.g. 0.5 to 15%
detergent.
[0058] To carry out the method of the invention, the isolated,
support-bound cells, may conveniently be removed or separated from
the remainder of the sample, thereby concentrating or enriching the
cells. Thus the leucocyte binding step serves to enrich the cells
or to concentrate them in a smaller volume than the initial sample.
To facilitate subsequent steps, it may be desirable, prior to the
lysis step, to dilute the support bound cells, e.g. in an
appropriate buffer or other medium. If desired the cells may
further be treated, e.g. by heating or mixing (e.g. vortexing). A
dilution step may be advantageous to prevent
agglomeration/aggregation of a particulate support such as beads,
particularly in a genomic DNA matrix which makes further handling
of the beads difficult and not reliable for transfer of the
supernatant or the bead pellet to another compartment or receptacle
e.g. well/tube/tray etc. Lysis then may conveniently be achieved by
adding an appropriate lysis buffer containing the desired lysis
agents or by subjecting the isolated cells to the desired lysis
conditions. For example, in the case of simply adding a lysis
buffer containing appropriate lysis agents, the isolated cells may
simply be incubated in the presence of the lysis buffer for a
suitable interval to allow lysis to take place. Different
incubation conditions may be appropriate for different lysis
systems, and are known in the art. For example for a detergent
containing lysis buffer, incubation may take place at room
temperature or at higher temperatures e.g. 37.degree. C.,
50.degree. C. or 65.degree. C. Likewise, time of incubation may be
varied from a few minutes e.g. 5 or 10 minutes to hours, e.g. 20 to
40 minutes or 1 to 2 hours. For enzymatic lysis, e.g. using
proteinase K etc, longer treatment times may be required, e.g.
overnight.
[0059] In an advantageous embodiment of the invention the lysis
step of the method comprises a further step involving the addition
of a further or extra amount of solid support to the isolated
leucocytes. Such "further" solid support (also referred to herein
as a "second" solid support) may comprise the same or a different
solid support from that used in step (a) of the method and may be
added to the cell sample as a separate component before or after
the addition of the lysis buffer or be included in the lysis
solution or buffer. The further or second solid support may thus
comprise any of the solid supports discussed above for use in step
(a). However, as the isolation of the leucocytes has already been
carried out by the lysis stage, there is no absolute requirement
for the second solid phase to have a leucocytes specific binding
partner associated with its surface. The use of a second solid
support has been found to offer advantages in sample collection for
example by improving pellet formation and hence isolation of the
first solid support. The improved pellet formation may also reduce
non-specific binding of substances or entities in the pellet, or in
other words reduces contamination of the pellet. Whilst not wishing
to be bound by theory it is believed that when only a first solid
support is used. the isolated nucleic acid binds to the first solid
support as a loose, non-compact mesh, thereby resulting in a
relatively loose non-compact pellet. However, where a second
support is used and particularly when this second solid support
comprises particles which are smaller or larger than the first
solid support, the second solid support fills in the gaps (or vice
versa) in the loose mesh, thereby making the pellet tighter and
more compact thus reducing the tendency to trap contaminating
material.
[0060] Thus the second solid support may be of comparable size and
density to the first solid support. Preferably however, the second
solid support is of a smaller size than the first solid support.
For example, where the supports are particulate the second solid
support comprises particles of a smaller diameter (e.g.
approximately half the diameter), than those comprising the first
solid support. In especially preferred embodiments the first solid
support comprises particles of 4.5 .mu.m diameter (e.g. the M450
beads described herein) whereas the second solid support comprises
particles of 2.8 .mu.m diameter (e.g. the M280 and M270 beads
described herein). Alternatively, the first support may be smaller
than the second support and the dimensions described above may be
reversed.
[0061] Especially preferably the second solid support may take a
more active role in the isolation of the nucleic acid and in such
cases the second solid phase is capable of binding to nucleic acid,
i.e. has nucleic acid binding properties. Preferably therefore the
second solid support may be made of glass, silica, latex, plastic
or any polymeric material (i.e. an uncoated surface) capable of
binding nucleic acid and such a solid support may optionally be
functionalised, for example to aid or improve nucleic acid binding.
Particularly preferred in this regard are functionalised solid
supports which have a surface charge, preferably a negative surface
charge. Most preferred are solid supports coated with carboxylic
acid functional groups. Such solid supports are commercially
available, for example the M-270 carboxylic acid beads or M-280
Hydroxyl beads manufactured by Dynal Biotech ASA. Preferably the
second solid supports are particulate, e.g. beads, and especially
preferably are magnetic.
[0062] The provision of a further or extra amount of solid support
after the cell isolation step results in an improved yield of
nucleic acid and also makes the elution of nucleic acid from the
solid support easier, particularly where solid supports with a
negatively charged surface are used. Whilst not wishing to be bound
by theory, as described above it is believed that the addition of
an extra amount of a "second" solid support improves the
compactness of the bead and nucleic acid pellet and particularly
where DNA is able to bind to the second solid support, more
effective and complete binding of nucleic acid molecules, rather
than the nucleic acid molecules being attached to the beads only at
one end or being attached to the beads loosely is achieved.
[0063] Thus, a further embodiment of the invention provides a
method of isolating nucleic acid from a blood sample, said method
comprising:
[0064] (a) selectively isolating leucocytes from said sample by
binding said leucocytes to a first solid support by means of a
binding partner specific for leucocytes;
[0065] (b) lysing said isolated leucocytes;
[0066] (c) binding nucleic acid released from said lysed cells to
said first solid support; and
[0067] (d) contacting said isolated leucocytes of step (b) or the
nucleic acid of step (c) with an additional amount of a second
solid support, preferably binding said nucleic acid to said second
solid support which is capable of binding nucleic acid.
[0068] Preferably the isolated lymphocytes are lysed in the
presence of said second solid support.
[0069] This method using a second solid support can equally be used
to selectively isolate nucleic acid from cells in any sample. In
such methods, the cells from which it is desired to isolate nucleic
acid are selectively isolated from the sample by binding said cells
to a first solid support by means of one or more appropriate
binding partners, after which steps (b), (c) and (d) of the method
described above are carried out on the particular isolated cells in
question.
[0070] Appropriate first and second solid supports for use in such
methods are discussed herein (and may be the same or different), as
are appropriate methods for the selective isolation of cells and
methods of lysis. Preferably the lysis step (b) of the method also
involves the use of proteinases and in particular proteinase K at
an appropriate concentration. As discussed above, the use of
proteinases in conventional techniques of nucleic acid isolation is
often disadvantageous since such materials tend to interfere with
many enzymic reactions and other downstream processing
applications. However, in preferred methods of the present
invention these disadvantageous effects of proteinases are
minimised by the use of magnetic separation technology wherein the
amount of contaminating enzymes will be negligible as the beads are
moved from vial to vial during the isolation of nucleic acid and
subsequent washing steps.
[0071] The terms "additional" or "extra" or "further" amount when
used herein in connection with the addition of a second solid
phase, is used to indicate the addition of any amount (by weight)
of second solid phase such that the isolation of nucleic acid is
improved, for example the yield of isolated nucleic acid is
improved. For example the amount of second solid phase added might
be the same amount as the amount of first solid phase used or may
be up to approximately 3 to 5 times the amount of first solid phase
used. Alternatively, the amount of second solid phase added may be
less than the amount of first solid phase providing that the
isolation of nucleic acid is improved. Preferably the amount of
second solid phase used is 0.5 to 3 times the amount of first solid
phase.
[0072] As the "amount" of solid phase refers to the weight of the
solid phase, in the preferred embodiments of the invention where
the first and second solid phases are particulate and the particles
making up the second solid phase are smaller (or larger) than the
particles making up the first solid phase, the number of particles
used for the first and second solid phases will generally be
different.
[0073] Following lysis, the released nucleic acid is bound to the
same support to which the lysed cells are bound or in other
embodiments of the invention the released nucleic acid is bound to
the same solid support to which the lysed cells are bound and the
additional second solid support. This nucleic acid binding may be
achieved in any way known in the art for binding nucleic acid to a
solid support. Conveniently, the nucleic acid is bound
non-specifically to the support i.e. independently of sequence.
Thus, for example the released nucleic acid may be precipitated
onto the support using any of the known precipitants for nucleic
acid, e.g. alcohols, alcohol/salt combinations, polyethylene
glycols (PEGs) etc. Precipitation of nucleic acids onto beads in
this manner is described for example in WO 91/12079. Thus, salt may
be added to the support and released nucleic acid in solution,
followed by addition of alcohol which will cause the nucleic acid
to precipitate. Alternatively, the salt and alcohol may be added
together, or the salt may be omitted. As described above in
relation to the cell binding step, any suitable alcohol or salt may
be used, and appropriate amounts or concentrations may readily be
determined. However, as mentioned above, it is preferred to avoid
the use of solvents, alcohols and similar agents. Thus alternative
techniques, avoiding the use of such agents are preferred.
[0074] One such alternative and preferred nucleic acid-binding
technique includes the use of detergents as described in WO
96/18731 of Dynal AS (the so-called "DNA Direct" procedure).
Various detergent-based systems for binding nucleic acids to a
solid support are described in this publication and may be used
according to the present invention.
[0075] Conveniently, the nucleic acid binding step may take place
simultaneously or concomitantly with the cell lysis step. This may
conveniently be achieved using the detergent-based methods of
WO96/18731. Thus, for example, an agent or agents for lysis and
nucleic acid binding may conveniently be contained in an
appropriate medium (e.g. a buffer or other aqueous solution) and
added to the support-bound cells. The cells may then be maintained
in contact with the medium e.g. incubated (e.g. as described above)
to allow lysis and nucleic acid binding to take place. Such a
medium may be referred to as a "lysis/binding" medium. A detergent
may function as both lysis agent and to assist in the binding of
the nucleic acid to the support.
[0076] The detergent may be any detergent, and a vast range are
known and described in the literature. Thus, the detergent may be
ionic, including anionic and cationic, non-ionic or zwitterionic.
The term "ionic detergent" as used herein includes any detergent
which is partly or wholly in ionic form when dissolved in water.
Anionic detergents have been shown to work particularly well and
are preferred. Suitable anionic detergents include for example
sodium dodecyl sulphate (SDS) or other alkali metal alkylsulphate
salts or similar detergents, sarkosyl, or combinations thereof.
[0077] Conveniently, the detergent may be used in a concentration
of 0.2 to 30% (w/v), e.g. 0.5 to 30%, preferably 0.5 to 15%, more
preferably 1 to 10%. For anionic detergents concentrations of 1.0
to 5% e.g. 0.5 to 5% have been shown to work well.
[0078] The detergent may be supplied in simple aqueous solution,
which may be alkaline or acidic, or more preferably in a buffer.
Any suitable buffer may be used, including for example Tris,
Bicine, Tricine, and phosphate buffers. Conveniently, a source of
monovalent cations, e.g. a salt, may be included to enhance nucleic
acid capture, although this is not necessary. Suitable salts
include chloride salts, e.g. sodium chloride, lithium chloride etc.
at concentrations of 0.1 to 1M, e.g. 250 to 500 mM. As mentioned
above, other components such as enzymes, may also be included.
[0079] Other optional components in the detergent composition
include chelating agents e.g. EDTA, EGTA and other polyamino
carboxylic acids conveniently at concentrations of 1 to 50 mM etc.,
reducing agents such as dithiotreitol (DTT) or
.beta.-mercaptoethanol, at concentrations of for example 1 to 10
mM.
[0080] Preferred detergent compositions may for example
comprise:
[0081] 100 mM Tris-HCl pH 7.5
[0082] 10 mM EDTA
[0083] 2% SDS
[0084] or:
[0085] 100 mM TrisCl pH 7.5
[0086] 10 mM EDTA
[0087] 5% SDS
[0088] 10 mM NaCl
[0089] or:
[0090] 100 mM TrisCl pH 7.5
[0091] 50 mM LiCl
[0092] 10 mM EDTA
[0093] 1% LiDS
[0094] Reference is made to WO96/18731 for further details,
exemplary reaction conditions etc.
[0095] In the embodiments of the invention where a second solid
support is added, this second solid support may be included in the
detergent composition. Further preferred detergent compositions
thus comprise the above compositions further comprising an
appropriate amount of second solid support, e.g. M270 carboxylic
acid beads or M-280 Hydroxyl beads, for example at a concentration
of approximately 1.5 mg/ml and optionally an appropriate amount of
proteinases, e.g. proteinase K, for example at 20 mg/ml.
[0096] By selecting appropriate "nucleic acid binding" conditions
(e.g. appropriate buffer or lysis/binding medium compositions), it
may be selected whether to bind DNA released from the cells, or RNA
released from the cells to the solid support. Thus, "binding
medium" compositions may be selected favouring DNA binding (or more
particularly genomic DNA binding) to the solid support. Such
binding medium compositions include those mentioned above, those
described in the Examples below, and the compositions of
WO96/18731. For example, a representative DNA binding medium may
include GuHCl and optionally EDTA.
[0097] To bind RNA, appropriate medium compositions or conditions
are known in the art, or may readily be determined from RNA
isolation procedures known in the art, and may include, for
example, the buffers and procedures described in EP-A-0389063 and
U.S. Pat. No. 5,234,809 of Akzo Nobel NV.
[0098] Representative RNA-binding compositions may thus include
guanidine thiocyanate (GTC) with EDTA.
[0099] For RNA binding, the nature of the solid support may be of
importance and in particular a "silica" (i.e. comprising silica
itself or being based on silica or a silica derivative) solid
surface should be used (see further below)
[0100] Advantageously, when the method of the invention is used to
isolate DNA, it may be combined with a further step separately to
isolate the RNA from the sample. Thus, following the procedure
discussed above, and selecting DNA-binding conditions in the
nucleic acid binding step (e.g. a lysis/binding or binding medium
favouring DNA binding), DNA released from the support-bound cells
may be bound to the support, and removed from the sample. RNA, most
notably mRNA, released from the leucocytes, remains in the sample
(more precisely in the supernatant). This RNA may readily be
isolated from the sample using standard procedures, for example by
binding to a capture probe, conveniently immobilised (e.g. by
binding to a solid support), consisting of oligo dT.
[0101] Alternative nucleic acid binding techniques may also be used
in order to achieve the step of binding released nucleic acid to
the solid support. For example, one such method may take advantage
of the well known principle of nucleic acid binding to a silica
surface.
[0102] Thus, in such an embodiment, the solid support may comprise
or consist of a silica or silica-based or derived material. Many
such materials are known and described in the art, and the
literature is replete with references describing the isolation of
nucleic acids by binding to silica surfaces (see e.g. EP-A-0389063
of AKZO N.V., U.S. Pat. No. 5,342,931, U.S. Pat. No. 5,503,816 and
U.S. Pat. No. 5,625,054 of Becton Dickinson, U.S. Pat. No.
5,155,018 of Hahnemann University, U.S. Pat. No. 6,027,945 of
Promega Corp. and U.S. Pat. No. 5,945,525 of Toyo Boseki KK).
[0103] Ionic binding of the nucleic acid to the support may be
achieved by using a solid support having a charged surface, for
example a support coated with polyamines.
[0104] The support which is used in the method of the invention may
also carry functional groups which assist in the specific or
non-specific binding of nucleic acids, for example DNA binding
proteins e.g. leucine zippers or histones or intercalating dyes
(e.g. ethidium bromide or Hoechst 42945) which may be coated onto
the support.
[0105] Likewise, the support may be provided with binding partners
to assist in the selective capture of nucleic acids. For example,
complementary DNA or RNA sequences, or DNA binding proteins may be
used. The attachment of such proteins to the solid support may be
achieved using techniques well known in the art. Conveniently, such
nucleic acid-binding partners may be intermixed on the solid
support with the anti-leucocyte binding partners.
[0106] Although not necessary, it may be convenient to introduce
one or more washing steps to the isolation method of the invention,
for example following the cell isolation and/or nucleic acid
binding step. Any conventional washing buffers or other media may
be used. Generally speaking, low to moderate ionic strength buffers
containing salt are preferred e.g. 10 mM Tris-HCl at pH 8.0/10 mM
or 40 mM NaCl. Other standard washing media, e.g. containing
alcohols, may also be used, if desired, for example washing with
70% ethanol.
[0107] The use of magnetic particles permits easy washing steps
simply by aggregating the particles, removing the nucleic acid
binding medium, adding the washing medium and reaggregating the
particles as many times as required.
[0108] Following the nucleic acid isolation process and any
optional washing steps which may be desired, the support carrying
the bound nucleic acid may be transferred e.g. resuspended or
immersed into any suitable medium e.g. water or low ionic strength
buffer. Thus, the isolated nucleic acid may be removed or separated
from the sample. Depending on the support and the nature of any
subsequent processing desired, it may or may not be desirable to
release the nucleic acid from the support.
[0109] In the case of a particulate solid support such as magnetic
or non-magnetic beads, this may in many cases be used directly, for
example in PCR or other amplifications, without eluting the nucleic
acid from the support. Also, for many DNA detection or
identification methods elution is not necessary since although the
DNA may be randomly in contact with the bead surface and bound at a
number of points by hydrogen bonding or ionic or other forces,
there will generally be sufficient lengths of DNA available for
hybridisation to oligonucleotides and for amplification.
[0110] However, if desired, elution of the nucleic acid may readily
be achieved using known means, for example by heating, e.g. to
65.degree. C. for 5 to 10 minutes, following which the support may
be removed from the medium leaving the nucleic acid in solution.
Such heating is automatically obtained in PCR by the DNA
denaturation step preceding the cycling program. An elution buffer
not containing salt may also conveniently be used.
[0111] If it is desired to remove RNA from DNA, this may be
achieved by destroying the RNA before the DNA separation step, for
example by addition of an RNAase or an alkali such as NaOH.
[0112] An advantage of the present invention, is that it is quick
and simple to perform, and with an appropriate combination of
cell-binding, lysis and nucleic acid binding steps, provides a
method which reliably and simply yields isolated nucleic acid in a
short period of time, in many cases, less than one hour, or even
less than 45 minutes. The simplicity of the method allows for high
throughput of samples. Concomitantly, the cell-binding step,
results in an enrichment or concentration of the cells, and
purification away from other components in the sample, thereby
improving the nucleic acid isolation process. Advantageously also,
the use of solvents such as chloroform/phenol is avoided.
Advantageously, the method of the invention permits nucleic acid to
be isolated from relatively small samples of blood, for example up
to 10 ml of blood, for example 10 .mu.l to 2 ml of blood, e.g. 200
to 500 .mu.l of blood or 50 to 200 .mu.l, (e.g. 100 .mu.l) of buffy
coat. The yields and quality of nucleic acid isolated using the
methods of the invention are good. For example 0.2 ml of blood
routinely provides 5-12 .mu.g DNA with an OD.sub.260/280 ratio of
1.75-1.9. In addition, as mentioned above, the methods of the
invention can be used to isolate nucleic acid from large samples of
blood, e.g. from samples of greater than 10 mls.
[0113] Particularly favourable results have been obtained using the
method of the invention to isolate genomic DNA from blood samples.
In particular, it has been shown that high quality DNA may be
obtained, with little fragmentation.
[0114] The invention is advantageously amenable to automation,
particularly if particles, and especially, magnetic particles are
used as the support. In a particularly favoured embodiment of the
invention, the nucleic acid isolation method is performed using an
automated system for handling of the solid support during the cell
lysis, nucleic acid binding, and, optionally, washing steps. Thus
the isolated support-bound cells may be transferred to such an
apparatus, washed if desired, and lysed; the nucleic acid may bind
to the support, and the bound nucleic acid may readily be washed,
using such an apparatus. Furthermore, such an apparatus may also be
used to handle the support during the cell isolation stage.
Particular mention may be made in this regard of the Bead
Retriever.TM., available from Dynal ASA, Norway. The apparatus has
a system for ready and efficient transfer of the support (carrying
cells or nucleic acid) from one well to another. Such an apparatus
is particularly efficacious in handling the high quality viscous
DNA which results from the method of the invention.
[0115] As mentioned above, the method of the invention has
particular utility as a preliminary first step to prepare nucleic
acid for use in nucleic acid-based detection procedures, for
example in genotyping.
[0116] As mentioned above, advantageously the bound nucleic acid
need not be eluted or removed from the support prior to carrying
out the detection step, although this may be performed if desired.
Whether or not the nucleic acid is eluted may also depend on the
particular method which was used in the nucleic acid binding step.
Thus certain nucleic acid-binding procedures will bind the nucleic
acid more tightly than others. In the case of DNA-binding using
detergents (e.g. by DNA Direct) for example, the nucleic acid will
elute from the solid support when an elution buffer or other
appropriate medium is introduced. Nucleic acid bound by means of a
precipitant such as alcohol or a chaotrope will remain more tightly
bound and may not elute when placed in a buffer medium, and may
require heating to be eluted.
[0117] Thus, the support-bound nucleic acid may be used directly in
a nucleic acid based detection procedure, especially if the support
is particulate, simply by resuspending the support in, or adding to
the support, a medium appropriate for the detection step. Either
the nucleic acid may elute into the medium, or as mentioned above,
it is not necessary for it to elute.
[0118] A number of different techniques for detecting nucleic acids
are known and described in the literature and any of these may be
used according to the present invention. Conveniently, nucleic acid
may be detected by optical methods, for example by measuring or
determining optical density (OD). Alternatively, the nucleic acid
may be detected by hybridisation to a probe and very many such
hybridisation protocols have been described (see e.g. Sambrook et
al., 1989, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold
Spring Harbor Press, Cold Spring Harbor, N.Y). Most commonly, the
detection will involve an in situ hybridisation step, and/or an in
vitro amplification step using any of the methods described in the
literature for this. Thus, as mentioned, techniques such as LAR,
3SR and the Q-beta-replicase system may be used. However, PCR and
its various modifications e.g. the use of nested primers, will
generally be the method of choice (see e.g. Abramson and Myers,
1993, Current Opinion in Biotechnology, 4: 41-47 for a review of
nucleic acid amplification technologies).
[0119] Other detection methods may be based on a sequencing
approach, for example, the minisequencing approach as described by
Syvnen and Soderlund, 1990, Genomics, 8: 684-692.
[0120] In amplification techniques such as PCR, the heating
required in the first step to melt the DNA duplex may release the
bound DNA from the support. Thus, in the case of a subsequent
detection step, such as PCR, the support bound nucleic acid may be
added directly to the reaction mix, and the nucleic acid will elute
in the first step of the detection process. The entire isolated
support bound nucleic acid sample obtained according to the
invention may be used in the detection step, or an aliquot.
[0121] The results of the PCR or other detection step may be
detected or visualised by many means, which are described in the
art. For example the PCR or other amplification products may be run
on an electrophoresis gel e.g. an ethidium bromide stained agarose
gel using known techniques. Alternatively, the DIANA system may be
used, which is a modification of the nested primer technique. In
the DIANA (Detection of Immobilised Amplified Nucleic Acids) system
(see Wahlberg et al., Mol. Cell Probes 4: 285(1990)), the inner,
second pair of primers carry, respectively, means for
immobilisation to permit capture of amplified DNA, and a label or
means for attachment of a label to permit recognition. This
provides the dual advantages of a reduced background signal, and a
rapid and easy means for detection of the amplified DNA.
[0122] The amplified nucleic acid may also be detected, or the
result confirmed, by sequencing, using any of the many different
sequencing technologies which are now available, e.g. standard
sequencing, solid phase sequencing, cyclic sequencing, automatic
sequencing and minisequencing.
[0123] Advantageously, it has been found that isolated cells may be
kept in a "cell-binding" buffer according to the invention e.g. a
salt/alcohol buffer for at least one week at room temperature with
no detectable loss of sensitivity in a subsequent nucleic acid
detection step. Such stability is an advantage in field
situations.
[0124] Thus, the methods of the invention may be used to isolate
nucleic acid for any appropriate subsequent use. Examples of such
uses are described briefly above. Advantageously the methods of the
invention could be used to prepare and isolate nucleic acid from
samples at the point of care, e.g. at or near the patient's
bed-side or in a doctor's surgery, where the nucleic acid isolated
or obtained could then optionally be used in down-stream testing
also at the point of care, e.g. could be used in yes/no gene
testing in for example a dot hybridization assay.
[0125] The various reactants and components required to perform the
methods of the invention may conveniently be supplied in kit form.
Such kits represent a further aspect of the invention.
[0126] At its simplest, this aspect of the invention provides a kit
for isolating nucleic acid from a sample comprising:
[0127] (a) a solid support;
[0128] (b) means for binding leucocytes to said solid support;
[0129] (c) means for lysing said cells; and
[0130] (d) means for binding nucleic acid released from said lysed
cells to said same solid support.
[0131] Further optional components of the kit include (e) a second
solid support, which may be the same or different to the solid
support component (a), and (f) a proteinase such as Proteinase K.
In the kits of the invention where a second solid support is
included it should be noted that such kits can equally be used to
isolate nucleic acid from any cell sample and are not limited to
isolating nucleic acid from a sample containing leucocytes, e.g. a
blood sample. In such kits, component (b) is replaced by an
appropriate means for binding the particular cells from which it is
desired to isolate nucleic acid to a solid support.
[0132] The various means (b), (c), (d), (e) and (f) may be as
described and discussed above, in relation to the methods of the
invention.
[0133] A further optional component is (g), means for detecting the
nucleic acid. As discussed above, such means may include
appropriate probe or primer oligonucleotide sequences for use in
hybridisation and/or amplification-based detection techniques.
[0134] Optionally further included in such a kit may be buffers,
salts, polymers, enzymes etc.
[0135] The invention will now be described in more detail in the
following non-limiting Examples with reference to the drawings in
which:
[0136] FIG. 1 is a chart showing yield of DNA (.mu.g) isolated from
a 100 .mu.l buffy coat sample as described in Example 2, isolations
I, II and III;
[0137] FIG. 2 is a chart showing yield of DNA (.mu.g) isolated from
a 100 .mu.l buffy coat sample as described in Example 3, isolations
I, II and III;
[0138] FIG. 3 is a chart showing % of DNA isolated from a 1 ml
blood sample as described in Example 4, using different
antibody-bead preparations (CD45/CD15 beads: KB 458-18; KB 458-16;
and KB 458-10; and separate CD45 and CD15 beads).
[0139] FIG. 4 is a graph showing the yield of DNA (.mu.g) isolated
from 200 .mu.l of blood as described in Example 6. The value of a
ratio giving an indication of DNA purity as described in Example 6
is also shown (.diamond-solid.).
EXAMPLE 1
[0140] General protocol for isolation of DNA from leucocytes
[0141] We have developed a method for isolation and purification of
DNA from specifically isolated cells, namely leucocytes, from
blood. The method is based on the use of antibodies or other
affinity molecules coated on a solid support, such as magnetic
beads for specific cell isolation. Subsequent lysis of these cells,
in an appropriate medium e.g. in a buffer with salt or chaotrope
containing a detergent, releases the DNA and the DNA adsorbs to the
beads. RNA and other contaminants remain in the supernatant and by
separation of the support, e.g. magnetically, the DNA/support
complex is washed to remove these residual contaminants. The DNA is
then resuspended in an appropriate buffer and is ready to be used
in downstream applications.
[0142] Leucocyte isolation
[0143] 375 .mu.g each of magnetic M450 CD45 and M450 CD15
Dynabeads.TM. (available from Dynal ASA) provided with anti-CD45 or
anti-CD15 antibody, (resuspended in PBS, pH 7.4, with 0.1% BSA and
0.02% NaN.sub.3) in a ratio of 1:1 are washed and resuspended in
DPBS (Dulbecco's PBS (phosphate buffered saline) used without
Ca.sup.2+ and Mg.sup.2+), pH 7.4, with 0.1% BSA and 0.6% NaCitrate.
The beads are then added to blood or buffy coat and incubated for
20-45 minutes at 4-20.degree. C. The cells are isolated
magnetically and washed in DPBS, pH 7.4, with 0.1% BSA and 0.6%
NaCitrate or added directly to Lysis/binding buffer (see Cell
lysis) without washing.
[0144] Cell lysis
[0145] The beads with the isolated cells attached added to
Lysis/Binding buffer (100 mM Tris-HCl, pH 7.5, 100 mM LiCl, 10 mM
EDTA, pH 8.0, 1% LiDS and 5 mM DTT (dithiothreitol)) and incubated
for five minutes at room temperature.
[0146] Washing and Elution
[0147] The DNA/bead complex is isolated magnetically and washed in
40 mM NaCl. The DNA is eluted in 10 mM Tris-HCl pH 7.4 by vigorous
pipetting and subsequent incubation at 65.degree. C. for 5 minutes.
The beads are magnetically removed from the eluted DNA.
EXAMPLE 2
[0148] Automated isolation of genomic DNA from buffy coat
[0149] In this procedure, both cell isolation and DNA isolation
steps are automated.
[0150] DNA isolation from 100 .mu.l buffy coat, diluted to 1 ml
with DPBS, pH 7.4, with 0.1% BSA and 0.6% NaCitrate, was performed
using 375 .mu.g M450 CD45 and 375 .mu.g M450 CD15. Both isolation
of cells and DNA was performed by using the Dynal BeadRetriever.TM.
apparatus. The isolated cells were transferred to Lysis/Binding
buffer and the DNA/bead complex was washed twice in 40 mM NaCl
before partial elution in 10 mM Tris-HCl pH 7.4. To elute the DNA
completely vigorous pipetting and elution at 65.degree. C. was
performed manually. The DNA yield was determined by measuring the
OD.sub.260 and OD.sub.280 and by using the Warburg-Christian
formula ([Nucleic Acid, .mu.g/ml]=62.9 OD.sub.260-36.0 OD.sub.280).
The purity of the isolated DNA was determined by the ratio
OD.sub.260/OD.sub.280 where a ratio between 1.7 and 2.0 is
considered as a pure DNA preparation. The results are shown in
Table 3 below, and also in FIG. 1.
3TABLE 1 Ratio Volume .mu.g/ml Parallel OD.sub.280 OD.sub.280
260/280 .mu.l DNA .mu.g/DNA I 0.758 0.457 1.66 170 31.23 5.31 II
0.778 0.456 1.71 189 32.56 6.15 III 0.792 0.459 1.73 180 33.29
5.99
EXAMPLE 3
[0151] Semi-automated isolation of genomic DNA for buffy coat
[0152] In this procedure, cells are isolated manually and DNA
isolation is automated.
[0153] DNA isolation from 100 .mu.l buffy coat, diluted to 300
.mu.l with DPBS, pH 7.4, with 0.1% BSA and 0.6% NaCitrate, was
performed using 375 .mu.g M450 CD45 and 375 .mu.g M450 CD15.
Isolation of cells was performed manually and DNA isolation was
performed by using the Dynal BeadRetriever.TM.. The manually
isolated cells were washed three times before addition of
Lysis/Binding buffer. The DNA/bead complex was washed three times
in 40 mM NaCl before partial elution in 10 mM Tris-HCl pH 7.4. To
elute the DNA completely vigorous pipetting and elution at
65.degree. C. was performed manually. The DNA yield was determined
by measuring the OD.sub.260 and OD.sub.280 and by using the
Warburg-Christian formula ([Nucleic Acid, .mu.g/ml]=62.9
OD.sub.260-36.0 OD.sub.280). The purity of the isolated DNA was
determined by the ratio OD.sub.260/OD.sub.280 where a ratio between
1.7 and 2.0 is considered as a pure DNA preparation. The results
are shown in Table 4 below, and also in FIG. 2.
4TABLE 4 Ratio Volume .mu.g/ml ug Parallel OD.sub.260 OD.sub.280
260/280 .mu.l DNA DNA I 0.942 0.522 1.80 186 40.46 7.53 II 0.834
0.463 1.80 205 35.79 7.34 III 1.104 0.624 1.77 180 46.98 8.46
EXAMPLE 4
[0154] DNA isolation from blood using beads with both CD45 and CD15
compared to beads with CD45 and CD15 on two different beads
[0155] Cells were isolated from 1 ml whole blood samples
(containing approximately 2.10.sup.7 leucocytes) using the general
procedure described in Example 1. Beads carrying both anti-CD45 and
anti-CD15 (KB 458-18; KB 458-16 and KB 458-10; these designations
represent different bead preparations) were compared to a procedure
using a combination of separate CD45 and CD15 beads (as in previous
Examples). 375 .mu.g of beads were used in each case.
[0156] The number of leucocytes in 1 ml of blood was determined by
flow cytometry to be 2,600,000. Blood samples were diluted from 9.6
ml to 12.5 ml, namely a 1.3.times. dilution, thereby resulting in
2,000,000 leucocytes. Assuming 5 pg DNA per cell gives an estimate
of DNA in 1 ml of blood of 12 .mu.g.
[0157] The results obtained are shown in Table 5 and in FIG. 3.
5 TABLE 5 .mu.g/ml ug % of Parallel OD.sub.260 OD.sub.280
OD.sub.320 Ratio Volume DNA DNA theoretical KB458- 1 0.766 0.471
0.081 1.76 157 29.05 4.6 38.00 18 2 1.117 0.752 0.155 1.61 180
39.02 7.0 58.53 3 1.334 0.901 0.21 1.63 180 45.82 8.2 68.74 KB458-
4 0.597 0.35 0.048 1.82 180 23.66 4.3 35.49 16 5 0.836 0.55 0.136
1.69 170 29.13 5.0 41.26 6 0.559 0.338 0.051 1.77 178 21.62 3.8
32.07 KB458- 7 1.216 0.815 0.185 1.64 182 42.17 7.7 63.96 10 8
1.161 0.736 0.142 1.72 190 42.17 8.1 67.63 9 0.00 0.0 0.00 CD45 10
0.934 0.581 0.09 1.72 175 35.41 6.2 51.64 CD15 11 1.054 0.654 0.096
1.72 181 40.17 7.3 60.59 12 0.961 0.587 0.082 1.74 185 37.11 6.9
57.21
[0158] No. WBC in 1 ml blood (determined by flow): 2,600,000
[0159] Blood diluted from 9.6 to 12.5 ml-1.3.times. diluted:
2,000,000
[0160] 6 pg DNA pr. cell gives ug DNA 1 ml blood=12
EXAMPLE 5
[0161] General Protocol for Isolation of DNA from leucocytes using
the Dynal BeadRetriever.TM.
[0162] Materials:
[0163] Blood
[0164] M450 CD45 and M450 CD15 or M450 CD45/15
[0165] DPBS/BSA (0.1% BSA og 0.6% NaCitrate) (50 ml DPBS+250 .mu.l
20% BSA+30 mg NaCitrate)
[0166] Lysis/Binding buffer (100 mM Tris-HCl, pH 7.5, 500 mM NaCl,
10 mM EDTA,
[0167] pH 8, 0.5 mM DTT, 1% SDS)
[0168] Washing buffer (40 mM NaCl)
[0169] 10 mM Tris-HCl ph 7.4
[0170] DPBS=Dulbecco's PBS used without Ca.sup.2+ and Mg.sup.2+
[0171] DTT=Dithiothreitol
[0172] Method:
[0173] Isolation and washing of leucocytes:
[0174] 1. Use 1.10.sup.7 beads per 1 ml blood. The beads are either
1:1 M450 CD45 and CD15 or M450 CD45/15.
[0175] 2. Remove the supernatant and wash the beads with DPBS/BSA
buffer.
[0176] 3. Add the beads to 2 ml tubes with screw cap.
[0177] 4. Add 1 ml blood. Mix carefully but well by pipetting.
[0178] 5. Incubate 20 min at 2-8.degree. C. on a roller.
[0179] 6. To the rack of tubes for BeadRetriever add:
[0180] Tube 1--empty (to add the 1 ml blood with isolated
cells)
[0181] Tube 2, 3, 4 and 5--1 ml DPBS/BSA
[0182] 7. After cell isolation, add the blood with the isolated
cells in the first tube of the BeadRetriever tube rack.
[0183] 8. Put the tubes and tips in the BeadRetriever and start the
program:
[0184] Tube 1--collect isolated cells
[0185] Tube 2, 3 and 4 wash 1 min
[0186] Tube 5--wash 1 min, collect cells on tips and put in
position 0
[0187] Lysis, binding of DNA and washing in BeadRetriever:
[0188] 9. To a new rack of tubes for BeadRetriever add:
[0189] Tube 1--500 .mu.l Lysis/Binding buffer
[0190] Tube 2, 3 and 4--1 ml Washing buffer
[0191] Tube 5--200 .mu.l 10 mM Tris-HCl pH 7.4
[0192] 10. Leave the tips in the machine, but replace the tubes
from cell washing with the new tubes for DNA binding and
washing.
[0193] 11. Start the program:
[0194] Tube 1--5 min lysis
[0195] Tube 2, 3 and 4--1 min washing
[0196] Tube 5--vigorous shaking to release the bead complex
[0197] Elution and determination of DNA purity and yield:
[0198] 12. DNA will not be released during shaking in the
BeadRetriever and needs to be handled manually. Transfer everything
in tube 5 to an Eppendorf tube and pipette up and down several
times until the beads are in solution.
[0199] 13. Elute the DNA at 65.degree. C. for 5 min. Transfer the
supernatant to a new tube.
[0200] 14. Determine the OD.sub.260, OD.sub.280 and OD.sub.320.
Calculate the ratio:
[0201] (OD.sub.280 and OD.sub.320)/(OD.sub.280 and OD.sub.320)
[0202] 15. Calculate the amount of DNA isolated:
[0203] .mu.g DNA =[62.9*(OD.sub.260 and
OD.sub.320)-36.0*(OD.sub.280 and OD.sub.320)]*ml elution volume
[0204] 16. Run 5 .mu.l sample on an agarose gel to visualise the
size an amount of DNA.
EXAMPLE 6
[0205] DNA isolation from leucocytes using both a first and a
second solid support
[0206] Material
[0207] Blood
[0208] M450 CD45 (4.times.10.sup.8 beads/ml and 30 mg/ml)
[0209] M450 CD15 (4.times.10.sup.8 beads/ml and 30 mg/ml)
[0210] DPBS with 0.1% BSA
[0211] Lysis/Binding buffer (100 mM Tris-HCl, pH 7.5, 500 mM LiCl,
10 mM EDTA, pH 8.0, 0.5 mM DTT, 1% LiDS) with 1.5 mg/ml
M270-COOH
[0212] 20 mg/ml Proteinase K
[0213] Washing buffer (10 mM Tris pH 8.0, 150 mM LiCl)
[0214] Resuspension buffer (Tris-HCl pH 8.0, 0.01% Tween-20)
[0215] Methods
[0216] Isolation and washing of leukocytes:
[0217] 1. Use 6.times.10.sup.6 beads (450 .mu.g) per 200 .mu.l
blood in a ratio of 2:1 of M450 CD45 and CD15. Remove the
supernatant and wash the beads with DPBS/BSA buffer.
[0218] 2. Add the washed beads to 200 .mu.l blood diluted in 200
.mu.l DPBS/BSA buffer. Mix carefully but well by pipetting.
[0219] 3. Incubate with constant movement for 20 minutes at room
temperature.
[0220] 4. Wash the isolated cells three times in DPBS/BSA buffer.
Change the tube at the first wash. Remove the supernatant.
[0221] Lysis of leukocytes and isolation of DNA
[0222] 5. Add 0.5 ml lysis/binding buffer containing 1.5 mg/ml
extra beads and 20 .mu.l of 20 mg/ml Proteinase K to the isolated
cells and beads. Do not mix by pipetting.
[0223] 6. Incubate for 5 minutes at room temperature with constant
movement.
[0224] 7. Wash three times by adding washing buffer without further
pipetting.
[0225] 8. Add 200 .mu.l resuspension buffer and incubate for 5
minutes at 80.degree. C.
[0226] 9. Spin shortly, pipette a few times and/or flick the tube.
Transfer the supernatant to a new tube.
[0227] Determination of DNA purity and yield:
[0228] 10. Determine the OD.sub.260, OD.sub.280, OD.sub.320.
Calculate the ratio:
(OD.sub.260-OD.sub.320)/(OD.sub.280-OD.sub.320)
[0229] 11. Calculate the amount of DNA isolated:
[0230] .mu.g DNA=[50.times.(OD.sub.260-OD.sub.320)].times.ml
elution volume.
[0231] The results from five different blood samples which have
been taken through this protocol are shown in FIG. 4.
* * * * *